Manipulating genomes Module 6 Flashcards
(43 cards)
3 techniques to study genes?
The polymerase chain reaction (PCR)
Gel electrophoresis
Cutting out DNA fragments, using restriction enzymes
How can multiple copies of a DNA fragment be made, using PCR?
A reaction mixture is set up, containing the DNA sample, free nucleotides, primers and DNA polymerase
DNA mixture is heated to 90 degrees, to break the hydrogen bonds between the 2 strands of DNA
Mixture is then cooled to between 50 and 65 degrees, so the primers can anneal (bind) to the strands
Reaction mixture heated to 72 degrees so DNA polymerase can work
DNA polymerase lines up free nucleotides with alongside each template strand, complementary base pairing means new complementary strands are formed
2 new copies of the fragment of DNA are formed, and one cycle of PCR is formed
What are primers?
Short pieces of DNA that are complementary to the bases at the start of the fragment you want
What’s DNA polymerase?
An enzyme that creates new DNA strands
Why is it useful that DNA polymerase doesn’t denature at high temperatures?
Useful as cycles of PCR can be carried out without having to use new enzyme each time
What happens when PCR is repeated?
Now have 4 strands (2 new and 2 original)
So next cycle there will be 8 then 16 then 32…
What is electrophoresis?
Procedure that uses an electrical current to separate out DNA fragments, RNA fragments, or proteins depending on their size
Explain electrophoresis steps
Row of wells is created at the end of some agrose gel, which is in a gel box/tank, with the wells nearest to the negative electrode
Add loading dye to each DNA sample, helps the sample sink to bottom of wells making them easier to see. Add a set amount of each DNA fragment solution to each well
Pass an electrical current through the gel, DNA fragments are negatively charged so will move towards the positive electrode.
Small DNA fragments will move faster and travel further, through the gel causing them to separate by size, turn power off when 2cm from end. Stain the DNA fragments
The bands of different DNA fragments will now be visible
How is electropheresis carried out on proteins?
Proteins can be positively or negatively charged, so before put in wells are mixed with a chemical that denatures the proteins so they all have the same charge
Used to identify blood or urine samples for disease
How can restriction enzymes be used to cut out DNA fragements?
Some sections of DNA have palindromic sequences of nucleotides, these sequences consist of anti-parallel base pairs (base pairs that read the same in the opposite direction)
Restriction enzymes are enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) the DNA at these places
Different restriction enzymes cut at different specific recognition sequences because the shape of the recognition sequence is complementary to an enzyme’s active site.
If recognition sequences are present at either side of the DNA you want, you can use restriction enzymes to separate it from the rest of the DNA
DNA sample is incubated with the specific restriction enzyme, which cuts the DNA fragment via a hydrolysis reaction
Sometimes cut leaves sticky ends (small tails of unpaired bases at each end of the fragment), as cut in a zig zag motion. Sticky ends can be used to anneal the DNA fragment to another piece of DNA, that has sticky ends with complementary sequences
What’s a genome?
All the genetic material in an organism
How can DNA profiles be created, and how do they distinguish between 2 different people?
Some of an organism’s genome contains repetitive non-coding base sequences
The number of times these repeat differ in person to person, therefore their length in nucleotides does aswell
The number of times a sequence is repeated (therefore the amount of nucleotides) at different loci (specific place), can be analysed using electrophoresis. Creating a DNA profile
Why is the probability having the same DNA profile very low?
Because having the same number of sequence repeats at each locus is very low
How can DNA profiling be used in forensic sciences?
DNA taken from all possible suspects
DNA is isolated
PCR is used to amplify multiple areas containing different sequence repeats
PCR products are run on electropheresis gel, and the profiles are compared to see if any match (have the same bands)
How DNA profilling be used to identify to see if samples have come from the same species?
The more similar the bands, the more likely they are from the same species
How can DNA profilling be used in medical diagnosis?
A DNA profile can refer to a unique pattern of several alleles
It can be used to analyse the risk of genetic disorders, where specific mutation is unknown or several mutations may have occurred, because it identifies a broader, altered genetic pattern
What is genetic engineering?
The manipulation of an Organism’s DNA
What’s a transformed organism?
Organisms that have had their DNA altered by genetic engineering, so have recombinant DNA (DNA formed by joining together DNA from different sources)
Brief outline of how genetic engineering process?
Involves extracting a gene from one organism, and then inserting it into another organism, normally of a different species. Or genes can also be manufactured instead of extracted from another organism.
The organism with the inserted gene will now produce the protein coded for by that gene
In depth description of how first step of genetic engineering works?
DNA fragment containing the desired genes is isolated using restriction enzymes
In depth explanation how the second step of Genetic engineering works?
The DNA fragment is inserted into a vector DNA (Vector is something used to transfer DNA into a cell, eg. plasmids (small circular molecules of DNA in bacteria, or bacteriophages (viruses that infect bacteria))
The vector DNA is cut open using the same restriction enzyme that was used to isolate the DNA fragment that containing the desired gene. So the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene
The vector DNA and DNA fragment are mixed together with DNA ligase
DNA ligase joins up the sugar-phosphate backbone of the 2 bits, = ligation
In depth explanation how the third step of genetic engineering works?
The vector with the recombinant DNA is then used to transfer the gene into the bacterial cells
If a plasmid vector is used, bacterial cells have to be convinced to take in the plasmid vector, and it’s DNA
Eg. suspension of bacterial cells is mixed with plasmid vector in a machine called an electroporator
The machine is switched on and an electrical field is created in the mixture, increasing the permeability of the bacterial cell membranes, and allows them to take in the plasmids, this is called electroporation
With a bacteriophage plasmid, the bacteriophage will infect the bacterium by injecting it’s DNA into it, the phage DNA then intergrates with the bacterial DNA
How can genetic engineering be used to improve plant yield?
Inserted with a gene that produces a protein that is toxic to some insects
Also positive ethical issue, as less chemical pesticides that farmers have to use on their crops
Negative ethical issues, promotes monoculture (only one type of crop planted, decreasing biodiversity, and leaves all crops vunerable to one disease as all genetically indentical
How can genetic engineering be used on animals?
They can be used to be make proteins, DNA fragments for protein injected into animal embryo, the embryo is then implanted into a female adult, when offspring is born, it’s tested to see if it can produce the protein. If it can it is reproduced using selective breeding
The protein is then extracted from the milk and put in the medicine
Positive ethical with pharming, is that drugs can be made in larger quantities, saving more people
Negative ethical issue, is that can harmful side effects o animals, and makes animals only assests
